A two-part research program has been designed to yield information about the structure and expression of the tRNA genes in E. coli and the effects of post-transcriptional modification on tRNA function. The specific objectives of the study are: 1) to determine the size, number and chromosomal distribution of the tRNA gene clusters in E. coli: 2) to determine if tRNA gene clusters are transcribed as polycistronic units; and 3) to identify the role(s) that specific modified nucleosides play in tRNA function. The size and number of the tRNA gene clusters will be determined from molecular weight and density measurements of purified 35S-tRNA:32P-DNA hybrids of different sizes. Preparations of purified tRNA cistrons will be used in the study. To determine if polycistronic tRNA operons occur in E. coli the RNA from cells conditionally defective in nucleolytic processing of precursor tRNA will be analyzed for the occurrence of multimeric tRNA precursors. The chromosomal location of the tRNA gene clusters will be determined by tRNA:DNA hybridization analyses with DNA from selected partial diploids of E. coli. Finally, the biological activities of 8 undermodified phenylalanine isoacceptor tRNAs and the fully modified species will be compared in an effort to reveal the role of tRNA base modification in the cell. The functions to be analyzed include: 1) aminoacylation, 2) ribosome binding, 3) coding response, 4) protein synthesis, 5) binding to the allosteric phenylalanine biosynthetic enzyme and 6) reactivity with phenylalanyl-tRNA-protein transferase.